EP3132262B1 - Gamma irradiation stabilized dextran solutions and methods of use - Google Patents
Gamma irradiation stabilized dextran solutions and methods of use Download PDFInfo
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- EP3132262B1 EP3132262B1 EP15716773.5A EP15716773A EP3132262B1 EP 3132262 B1 EP3132262 B1 EP 3132262B1 EP 15716773 A EP15716773 A EP 15716773A EP 3132262 B1 EP3132262 B1 EP 3132262B1
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- dextran
- solution
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- 229920002307 Dextran Polymers 0.000 title claims description 108
- 238000000034 method Methods 0.000 title claims description 31
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 claims description 48
- 239000000243 solution Substances 0.000 claims description 40
- 210000003743 erythrocyte Anatomy 0.000 claims description 29
- 235000010323 ascorbic acid Nutrition 0.000 claims description 24
- 239000011668 ascorbic acid Substances 0.000 claims description 24
- 150000003839 salts Chemical class 0.000 claims description 21
- 229960005070 ascorbic acid Drugs 0.000 claims description 17
- 229910052500 inorganic mineral Inorganic materials 0.000 claims description 17
- 239000011707 mineral Substances 0.000 claims description 17
- 239000000872 buffer Substances 0.000 claims description 10
- 239000007864 aqueous solution Substances 0.000 claims description 9
- 210000004027 cell Anatomy 0.000 claims description 9
- 239000003623 enhancer Substances 0.000 claims description 9
- 231100000252 nontoxic Toxicity 0.000 claims description 9
- 230000003000 nontoxic effect Effects 0.000 claims description 9
- 239000001509 sodium citrate Substances 0.000 claims description 8
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical group O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 claims description 8
- 230000005855 radiation Effects 0.000 claims description 7
- 235000010378 sodium ascorbate Nutrition 0.000 claims description 7
- PPASLZSBLFJQEF-RKJRWTFHSA-M sodium ascorbate Substances [Na+].OC[C@@H](O)[C@H]1OC(=O)C(O)=C1[O-] PPASLZSBLFJQEF-RKJRWTFHSA-M 0.000 claims description 7
- 229960005055 sodium ascorbate Drugs 0.000 claims description 7
- PPASLZSBLFJQEF-RXSVEWSESA-M sodium-L-ascorbate Chemical group [Na+].OC[C@H](O)[C@H]1OC(=O)C(O)=C1[O-] PPASLZSBLFJQEF-RXSVEWSESA-M 0.000 claims description 7
- 239000013049 sediment Substances 0.000 claims description 5
- 229940074404 sodium succinate Drugs 0.000 claims description 5
- ZDQYSKICYIVCPN-UHFFFAOYSA-L sodium succinate (anhydrous) Chemical compound [Na+].[Na+].[O-]C(=O)CCC([O-])=O ZDQYSKICYIVCPN-UHFFFAOYSA-L 0.000 claims description 5
- 239000007791 liquid phase Substances 0.000 claims description 4
- 238000005119 centrifugation Methods 0.000 claims description 2
- 238000005374 membrane filtration Methods 0.000 claims description 2
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical group C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims 2
- 239000011734 sodium Substances 0.000 claims 2
- 229910052708 sodium Inorganic materials 0.000 claims 2
- 210000000677 aggregate cell Anatomy 0.000 claims 1
- 239000000523 sample Substances 0.000 description 16
- 238000004062 sedimentation Methods 0.000 description 10
- 229940072107 ascorbate Drugs 0.000 description 7
- 210000004369 blood Anatomy 0.000 description 7
- 239000008280 blood Substances 0.000 description 7
- 239000000654 additive Substances 0.000 description 5
- 210000000601 blood cell Anatomy 0.000 description 5
- 210000004700 fetal blood Anatomy 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 4
- 239000000306 component Substances 0.000 description 4
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 4
- 238000005227 gel permeation chromatography Methods 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 238000000569 multi-angle light scattering Methods 0.000 description 4
- 239000002953 phosphate buffered saline Substances 0.000 description 4
- 239000000376 reactant Substances 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 3
- 239000000561 aggregant Substances 0.000 description 3
- 230000002776 aggregation Effects 0.000 description 3
- 238000004220 aggregation Methods 0.000 description 3
- VZTDIZULWFCMLS-UHFFFAOYSA-N ammonium formate Chemical compound [NH4+].[O-]C=O VZTDIZULWFCMLS-UHFFFAOYSA-N 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 230000001954 sterilising effect Effects 0.000 description 3
- 238000004659 sterilization and disinfection Methods 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 230000004931 aggregating effect Effects 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 238000004364 calculation method Methods 0.000 description 2
- 238000013354 cell banking Methods 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 238000010829 isocratic elution Methods 0.000 description 2
- 230000007774 longterm Effects 0.000 description 2
- 210000005259 peripheral blood Anatomy 0.000 description 2
- 239000011886 peripheral blood Substances 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 230000001172 regenerating effect Effects 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 238000002054 transplantation Methods 0.000 description 2
- GUTLYIVDDKVIGB-OUBTZVSYSA-N Cobalt-60 Chemical compound [60Co] GUTLYIVDDKVIGB-OUBTZVSYSA-N 0.000 description 1
- 125000003535 D-glucopyranosyl group Chemical group [H]OC([H])([H])[C@@]1([H])OC([H])(*)[C@]([H])(O[H])[C@@]([H])(O[H])[C@]1([H])O[H] 0.000 description 1
- 239000004260 Potassium ascorbate Substances 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000012491 analyte Substances 0.000 description 1
- 239000008346 aqueous phase Substances 0.000 description 1
- 238000000149 argon plasma sintering Methods 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 239000012503 blood component Substances 0.000 description 1
- 210000001185 bone marrow Anatomy 0.000 description 1
- 235000010376 calcium ascorbate Nutrition 0.000 description 1
- 229940047036 calcium ascorbate Drugs 0.000 description 1
- 239000011692 calcium ascorbate Substances 0.000 description 1
- BLORRZQTHNGFTI-ZZMNMWMASA-L calcium-L-ascorbate Chemical compound [Ca+2].OC[C@H](O)[C@H]1OC(=O)C(O)=C1[O-].OC[C@H](O)[C@H]1OC(=O)C(O)=C1[O-] BLORRZQTHNGFTI-ZZMNMWMASA-L 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 239000013068 control sample Substances 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000011038 discontinuous diafiltration by volume reduction Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 230000005484 gravity Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 229940074358 magnesium ascorbate Drugs 0.000 description 1
- AIOKQVJVNPDJKA-ZZMNMWMASA-L magnesium;(2r)-2-[(1s)-1,2-dihydroxyethyl]-4-hydroxy-5-oxo-2h-furan-3-olate Chemical compound [Mg+2].OC[C@H](O)[C@H]1OC(=O)C(O)=C1[O-].OC[C@H](O)[C@H]1OC(=O)C(O)=C1[O-] AIOKQVJVNPDJKA-ZZMNMWMASA-L 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 235000019275 potassium ascorbate Nutrition 0.000 description 1
- 229940017794 potassium ascorbate Drugs 0.000 description 1
- CONVKSGEGAVTMB-RXSVEWSESA-M potassium-L-ascorbate Chemical compound [K+].OC[C@H](O)[C@H]1OC(=O)C(O)=C1[O-] CONVKSGEGAVTMB-RXSVEWSESA-M 0.000 description 1
- 238000001448 refractive index detection Methods 0.000 description 1
- 159000000000 sodium salts Chemical class 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 238000001370 static light scattering Methods 0.000 description 1
- 210000000130 stem cell Anatomy 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 239000008215 water for injection Substances 0.000 description 1
- 229940056904 zinc ascorbate Drugs 0.000 description 1
- WWRJFSIRMWUMAE-ZZMNMWMASA-L zinc;(2r)-2-[(1s)-1,2-dihydroxyethyl]-3-hydroxy-5-oxo-2h-furan-4-olate Chemical compound [Zn+2].OC[C@H](O)[C@H]1OC(=O)C(O)=C1[O-].OC[C@H](O)[C@H]1OC(=O)C(O)=C1[O-] WWRJFSIRMWUMAE-ZZMNMWMASA-L 0.000 description 1
Images
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/0081—Purging biological preparations of unwanted cells
- C12N5/0087—Purging against subsets of blood cells, e.g. purging alloreactive T cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/50—Soluble polymers, e.g. polyethyleneglycol [PEG]
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/483—Physical analysis of biological material
- G01N33/487—Physical analysis of biological material of liquid biological material
- G01N33/49—Blood
- G01N33/491—Blood by separating the blood components
Definitions
- red blood cells red blood cells
- dextran as an aggregant to enhance the sedimentation of red blood cells from whole blood.
- a critical part of the manufacturing process is the sterilization of the kit which is accomplished by exposure to gamma irradiation at a dose between 20 and 50 kGy, sufficient to ensure sterilization.
- dextran in water is unstable to gamma irradiation resulting in severe molecular weight decomposition of the dextran. For example, 3,300 kD Mw dextran will decompose to less than 20 kD on exposure to only a 20 kGy dose of gamma irradiation.
- RBC sedimentation enhancement performance of the added dextran is a function of its molecular weight and is ineffective below 200 kD molecular weight.
- the dextran solution must be sterilized separately by autoclaving or filtering the solution then reassembling with the gamma sterilized kit adding cost and potential contamination during manufacturing.
- US 2003/109494 discloses a solution comprising Dextran (2 grams, average molecular weight 15,000) dissolved in water (100 ml) at pH 5.2 to which Ascorbic acid (2 grams) was added.
- US2009/081689 discloses a method of aggregating cells in a sample containing red blood cells involving adding a dextran solution with dextrans of over 500kD.
- the methods and kits of the invention provide gamma stable dextran solutions, which can be sterilized through gamma irradiation while maintaining a sufficient molecular weight to subsequently act as a red blood cell (RBC) aggregant.
- RBC red blood cell
- One embodiment provides an aqueous dextran solution, stable to gamma irradiation.
- the solutions comprising 1 to 10 wt /v % of dextran, where the dextran has an initial average molecular weight greater than 500kD, and 2.0 to 20.0 wt% ascorbic acid or its mineral salt to the dextran.
- a kit for producing a gamma stabilized aqueous dextran solution comprising dextran having an initial average molecular weight greater than 500kD, and 2.0 to 20.0 wt% ascorbic acid or its mineral salt to the dextran.
- a method provides for adding the gamma stabilized aqueous solution to a blood sample (peripheral blood, cord blood), resulting in increased red blood cells (RBC) aggregation and sedimentation while recovering a large percentage of the total nucleated cells (TNC).
- the method comprises the steps of subjecting a dextran solution comprising ascorbic acid or a mineral salt of ascorbic acid to gamma radiation, adding to the blood sample, incubating to aggregate RBCs, and recover TNCs.
- “Dextran” refers to polysaccharides with molecular weights ⁇ 1000 Dalton (Da), which have a linear backbone of ⁇ -linked D-glucopyranosyl repeating units and typically have a molecular weight ranging from 3,000Da to 2,000,000Da. It is often classified according to molecular weight. For example dextran 500 refers to an average molecular mass of 500 kDa. Dextran 1230 refers to an average molecular mass of 1230kDa.
- Kit is referred to herein as one or more reactants or additives necessary for a given assay, test, or process.
- the kit may also include a set of directions to use the reactants or additives present in the kit, any buffers necessary to maintain processing conditions or other optional materials for using. In certain cases the kit may contain premeasured amounts of the reactants or additives for a given assay, test, or process.
- a gamma stabilized dextran solution comprising of an aqueous solution of dextran, ascorbic acid or a mineral salt of ascorbic acid.
- the dextran solution is approximately 1 to 10 wt% of dextran in an aqueous solution.
- the dextran is 1 to 5wt % and more preferable 2 to 4wt % dextran in an aqueous solution.
- the red blood cell sedimentation enhancement performance of added dextran is a function of molecular weight and it is ineffective below 200kD molecular weight.
- the dextran after gamma irradiation the dextran has a molecular weight greater than approximately 200kD.
- the molecular weight is in a range of approximately 200-800 kD and most preferred the dextran has molecular weight in a range of approximately 400-600 kD after gamma irradiation.
- the dose of gamma radiation is between 20 and 50kGy, and more preferably at a dose between 25 and 45 kGy.
- the ascorbic acid is a mineral salt including, but not limited to sodium ascorbate, calcium ascorbate, potassium ascorbate, magnesium ascorbate, zinc ascorbate or a combination thereof.
- the mineral salt is sodium ascorbate.
- the ascorbic acid or its mineral salt added from approximately 2 to 20 wt% to an aqueous dextran in preferred embodiments from 4 to 15 wt %, and more preferable from 4 to 10 wt %.
- the mineral salt is added directly to dextran at approximately 2 to 20 wt%, in preferred embodiments from 4 to 15 wt %, and more preferable from 4 to 10 wt%.
- the ascorbic acid or its mineral salt may act as a radiation stabilizer, conserving the dextran molecular weight at a level sufficient to act as an aggregant to enhance the sedimentation of RBC.
- the gamma stabilized dextran solution may further comprise buffers.
- the buffer comprises organic or inorganic salts that maintain a pH of 4.0 to 8.0 such as such as phosphate buffered saline (PBS).
- PBS phosphate buffered saline
- the solution may also comprise other non-toxic enhancers such as, sodium citrate, sodium succinate and combinations thereof.
- non-toxic enhancers the methods of aggregating blood cells and its use in connection with the system and methods are further described in U.S. Patent Application, Serial No. 12/325672 , entitled "SYSTEMS AND METHODS FOR PROCESSING COMPLEX BIOLOGICAL MATERIALS'.
- a method to sediment cells improve the resulting recovery of an increased percentage of total nucleated cells (TNCs) from a sample comprising red blood cells (RBC), where the method comprises the steps of adding the RBC sample to the aforementioned gamma stabilized dextran solution comprising ascorbic acid or a mineral salt of ascorbic acid to (Step A).
- the method further comprises incubation of the sample to aggregate and sediment the plurality of RBCs (Step C) and/or eventual recovering the TNC (Step D).
- Steps C and D are commonly performed in long-term cell banking and regenerative medicinal applications, where a maximal yield of nucleated blood cells is desired in a reduced volume for direct transplantation or storage for future use (Step D).
- the method of recovering the TNC may comprise concentration of a liquid phase prior to collection.
- the liquid phase comprises plasma and dextran and thus concentration may be accomplished through a number of methods including centrifugation, membrane filtration, or a combination of methods.
- the sample comprising the RBC is whole blood
- the sample comprising the RBC is a blood component, such as but not limited to isolated blood fraction including bone marrow and mobilized peripheral blood.
- a kit comprising the reactants and additives necessary for producing a gamma stabilized dextran solution.
- the kit comprises of dextran, abscorbic acid or a mineral salt of ascorbic acid.
- the dextran has a molecular weight (MW) that is sufficient to maintain a MW greater than approximately 200kD after exposure to gamma radiation.
- the initial molecular weight of dextran is greater than 500 kD, in preferred embodiments greater than 750kD and most preferred greater than 1000kD.
- the initial molecular weight of dextran is between 1000kD and 2000kD, in preferred embodiments the initial molecular weight of dextran is approximately 1000-1500kD.
- the components of the kit are dry mixed in an amount that, when added to an aqueous solution, yields a gamma stabilized dextran solution of approximately 1 to 10 wt% of dextran in an aqueous solution.
- the dextran is 1 to 5wt % and more preferable 2 to 4wt % dextran when used in an aqueous solution.
- the kit may further comprise buffers such as phosphate buffered saline (PBS), saline and or other non-toxic enhancers such as, sodium citrate, sodium succinate and combinations thereof.
- PBS phosphate buffered saline
- the additives may be combined in such a way that when added together in an aqueous solution, a gamma stabilized dextran solution is obtained.
- the kit is prepared in such a way that the individual components are provided separately.
- the kit is prepared such that components in solid form may be premixed and supplied together.
- the kit is prepared such that certain components are provided in solution.
- the methods and kits of the invention to sediment blood cells generally comprise adding the gamma stabilized dextran solution to accelerate RBC sedimentation.
- a sample that includes red blood cells is treated by adding an irradiated gamma stabilized dextran solution, followed by incubation of the sample, and eventual recovery of the total nucleated cells (TNCs).
- TMCs total nucleated cells
- One or more of the methods of recovering a percentage of TNCs from a sample comprising red blood cells comprises adding a gamma stabilized dextran solution which has been irradiated, at a predetermined concentration,, incubation of the sample, and eventually recovering of the total nucleated cells.
- the dextran 1230 used in this example was obtained from GE Healthcare, (Uppsala, Sweden). Ammonium formate and sodium ascorbate are available from Sigma-Aldrich (St. Louis, Missouri). Sodium citrate was obtained from Thermo Fisher Scientific (Waltham, Massachusetts). Pure dextran samples were prepared by dissolving approximately 37.5mM of sodium citrate and 2.25% (w/v) of dextran in 40% (v/v) of saline (Baxter, Deerfield IL) and 31.6 % (v/v) of water for injection (Baxter, IL). Dextran and sodium citrate were allowed to dissolve for 2 hours after which the remaining saline was added to make up the desired volume. All samples were allowed to dissolve for at least 2hr prior to analysis.
- Static Light Scattering analysis in combination with aqueous phase gel permeation chromatography was achieved using an Agilent 1100 series HPLC in combination with a Wyatt Technologies Dawn EOS Multi-Angle Light Scattering Detector in-line to a Wyatt Optilab DSP Interferometric Refractive index detector.
- the light scattering detector collects scattered light at 18 angles upstream of the refractive index (DRI) data acquisition.
- the function of the DRI detector is to provide a concentration term from the RI response of a given analyte with a known refractive index increment value (DN/DC) for use in the first principle calculation equation for molar mass.
- the chromatography was achieved using an isocratic elution of 2mM ammonium formate (pH 4-5). The molar mass values were obtained using the first principle calculation as derived from the Zimm formalism.
- the GPC separation was achieved using 2 Tosoh PW Columns: 1-G6000 and 1-G3000 aqueous SEC columns (7.5x300mm) in series at a flow rate of 1ml min-1 run at ambient temperature as outlined in Table 1.
- Table 1 Method Parameters for Gel Permeation Chromatography in combination with Multi-Angle Laser Light Scattering / Differential Refractive Index Detection of Dextrans.
- Table 2 shows the effects of ascorbic acid or its sodium salt added from 1.0 to 10.0 wt% to an aqueous dextran solution followed by exposure to 40 kGy dose of Gamma irradiation (Cobalt 60) Table 2.
- Dextran solutions exposed to 40kGy dose of gamma radiation Sample # Dextran Mw (kD) % Ascorbate Gamma dose (kGy) Mw after irradiation 6
- Dextran AB (1,230) 0 none 1,230 12 Dextran AB (1,230) 0 40 35 13 Dextran AB (1,230) 1 40 51 14 Dextran AB (1,230) 2 40 95 13 Dextran AB (1,230) 4 40 342 14 Dextran AB (1,230) 5 40 413 15 Dextran AB (1,230) 6 40 452 16 Dextran AB (1,230) 10 40 614
- Dextran having a Mw of greater than 200 kD is desirable for optimal sedimentation performance. Using combination of starting high molecular weight dextran in the presence of ascorbate achieves that goal. However due to the presence of ascorbate it was still desirable to confirm the cord blood sedimentation performance.
- red blood cell aggregation was measured in vitro by mixing 5 mls cord blood (sourced from New York Blood Center) in a 20 ml tube (from Globe Scientific) with 10mls of aqueous dextran solutions from Table 4. A control sample was also prepared without the sodium ascorbate. The tube was capped and the contents mixed well by inverting the tube up and down. The contents were allowed to settle under gravity and the height of the RBC pellet was measured at 0,10,20,40, and 60 minutes.
- Table 4 shows that gamma irradiated dextran's in the presence of sodium ascorbate aggregates RBCs to similar extents as non-irradiated dextran's with and without sodium ascorbate.
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Description
- Separation of red blood cells (RBC) from whole blood is commonly required prior to analysis or therapeutic use of less abundant cells, such as white blood cells or stem cells. Many conventional blood cell isolation procedures require preliminary red blood cell depletion and sample volume reduction. These steps are commonly performed in long-term cell banking and regenerative medicinal applications, where a maximal yield of nucleated blood cells is desired in a reduced volume for direct transplantation or storage for future use.
- Often these methods utilize dextran as an aggregant to enhance the sedimentation of red blood cells from whole blood. A critical part of the manufacturing process is the sterilization of the kit which is accomplished by exposure to gamma irradiation at a dose between 20 and 50 kGy, sufficient to ensure sterilization. Unfortunately, dextran in water is unstable to gamma irradiation resulting in severe molecular weight decomposition of the dextran. For example, 3,300 kD Mw dextran will decompose to less than 20 kD on exposure to only a 20 kGy dose of gamma irradiation. Furthermore, RBC sedimentation enhancement performance of the added dextran is a function of its molecular weight and is ineffective below 200 kD molecular weight. As a result the dextran solution must be sterilized separately by autoclaving or filtering the solution then reassembling with the gamma sterilized kit adding cost and potential contamination during manufacturing.
US 2003/109494 discloses a solution comprising Dextran (2 grams, average molecular weight 15,000) dissolved in water (100 ml) at pH 5.2 to which Ascorbic acid (2 grams) was added. -
US2009/081689 discloses a method of aggregating cells in a sample containing red blood cells involving adding a dextran solution with dextrans of over 500kD. - As such there is a need for gamma stabilized dextran solutions which will allow incorporation of the dextran more directly into the handling and manufacturing process to insure stability but also reduce handling errors and cost.
- In general, the methods and kits of the invention provide gamma stable dextran solutions, which can be sterilized through gamma irradiation while maintaining a sufficient molecular weight to subsequently act as a red blood cell (RBC) aggregant. This increases the efficiency of blood separation as the dextran solution may be incorporated directly into the handling and manufacturing process
- One embodiment provides an aqueous dextran solution, stable to gamma irradiation. The solutions comprising 1 to 10 wt /v % of dextran, where the dextran has an initial average molecular weight greater than 500kD, and 2.0 to 20.0 wt% ascorbic acid or its mineral salt to the dextran.
- In another embodiment, a kit is provided for producing a gamma stabilized aqueous dextran solution comprising dextran having an initial average molecular weight greater than 500kD, and 2.0 to 20.0 wt% ascorbic acid or its mineral salt to the dextran.
- In another embodiment a method provides for adding the gamma stabilized aqueous solution to a blood sample (peripheral blood, cord blood), resulting in increased red blood cells (RBC) aggregation and sedimentation while recovering a large percentage of the total nucleated cells (TNC). The method comprises the steps of subjecting a dextran solution comprising ascorbic acid or a mineral salt of ascorbic acid to gamma radiation, adding to the blood sample, incubating to aggregate RBCs, and recover TNCs.
- These and other features, aspects, and advantages of the present invention will become better understood when the following detailed description is read with reference to the accompanying figure.
-
FIG. 1 is flow diagram of the process to sediment cells using a gamma stabilized dextran solution which undergoes sterilization through exposure to gamma irradiation. - The following detailed description is exemplary and not intended to limit the invention of the application and uses of the invention. Furthermore, there is no intention to be limited by any theory presented in the preceding background of the invention on the following detailed description. To more clearly and concisely describe and point out the subject matter of the claimed invention, the following definitions are provided for specific terms that are used in the following description and the claims appended hereto.
- Unless otherwise indicated, the article "a" refers to one or more than one of the word modified by the article "a." Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, so forth used in the specification and claims are to be understood as being modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.
- "Dextran" refers to polysaccharides with molecular weights ≥1000 Dalton (Da), which have a linear backbone of α-linked D-glucopyranosyl repeating units and typically have a molecular weight ranging from 3,000Da to 2,000,000Da. It is often classified according to molecular weight. For example dextran 500 refers to an average molecular mass of 500 kDa. Dextran 1230 refers to an average molecular mass of 1230kDa.
- "Kit" is referred to herein as one or more reactants or additives necessary for a given assay, test, or process. The kit may also include a set of directions to use the reactants or additives present in the kit, any buffers necessary to maintain processing conditions or other optional materials for using. In certain cases the kit may contain premeasured amounts of the reactants or additives for a given assay, test, or process.
- In certain embodiments a gamma stabilized dextran solution is provided, the solution comprising of an aqueous solution of dextran, ascorbic acid or a mineral salt of ascorbic acid. In certain embodiments, the dextran solution is approximately 1 to 10 wt% of dextran in an aqueous solution. Preferably the dextran is 1 to 5wt % and more preferable 2 to 4wt % dextran in an aqueous solution. In certain embodiments the red blood cell sedimentation enhancement performance of added dextran is a function of molecular weight and it is ineffective below 200kD molecular weight. As such in certain embodiments, after gamma irradiation the dextran has a molecular weight greater than approximately 200kD. In preferred embodiments, the molecular weight is in a range of approximately 200-800 kD and most preferred the dextran has molecular weight in a range of approximately 400-600 kD after gamma irradiation. In certain embodiments the dose of gamma radiation is between 20 and 50kGy, and more preferably at a dose between 25 and 45 kGy.
- In certain embodiments, the ascorbic acid is a mineral salt including, but not limited to sodium ascorbate, calcium ascorbate, potassium ascorbate, magnesium ascorbate, zinc ascorbate or a combination thereof. In certain embodiments the mineral salt is sodium ascorbate.
- In certain embodiments the ascorbic acid or its mineral salt added from approximately 2 to 20 wt% to an aqueous dextran in preferred embodiments from 4 to 15 wt %, and more preferable from 4 to 10 wt %. In certain other embodiments the mineral salt is added directly to dextran at approximately 2 to 20 wt%, in preferred embodiments from 4 to 15 wt %, and more preferable from 4 to 10 wt%. The ascorbic acid or its mineral salt may act as a radiation stabilizer, conserving the dextran molecular weight at a level sufficient to act as an aggregant to enhance the sedimentation of RBC.
- In certain embodiments, the gamma stabilized dextran solution may further comprise buffers. The buffer comprises organic or inorganic salts that maintain a pH of 4.0 to 8.0 such as such as phosphate buffered saline (PBS). The solution may also comprise other non-toxic enhancers such as, sodium citrate, sodium succinate and combinations thereof. The use of non-toxic enhancers, the methods of aggregating blood cells and its use in connection with the system and methods are further described in
U.S. Patent Application, Serial No. 12/325672 , entitled "SYSTEMS AND METHODS FOR PROCESSING COMPLEX BIOLOGICAL MATERIALS'. - As shown in
FIG. 1 in certain embodiments a method to sediment cells improve the resulting recovery of an increased percentage of total nucleated cells (TNCs) from a sample comprising red blood cells (RBC), where the method comprises the steps of adding the RBC sample to the aforementioned gamma stabilized dextran solution comprising ascorbic acid or a mineral salt of ascorbic acid to (Step A). In certain embodiments, the method further comprises incubation of the sample to aggregate and sediment the plurality of RBCs (Step C) and/or eventual recovering the TNC (Step D). For example, Steps C and D are commonly performed in long-term cell banking and regenerative medicinal applications, where a maximal yield of nucleated blood cells is desired in a reduced volume for direct transplantation or storage for future use (Step D). In certain embodiments, the method of recovering the TNC may comprise concentration of a liquid phase prior to collection. The liquid phase comprises plasma and dextran and thus concentration may be accomplished through a number of methods including centrifugation, membrane filtration, or a combination of methods. - In certain embodiments the sample comprising the RBC is whole blood, in certain other embodiments the sample comprising the RBC is a blood component, such as but not limited to isolated blood fraction including bone marrow and mobilized peripheral blood.
- In certain embodiments, a kit is provided comprising the reactants and additives necessary for producing a gamma stabilized dextran solution. In certain embodiments, the kit comprises of dextran, abscorbic acid or a mineral salt of ascorbic acid. In certain embodiments, the dextran has a molecular weight (MW) that is sufficient to maintain a MW greater than approximately 200kD after exposure to gamma radiation. In certain embodiments, the initial molecular weight of dextran is greater than 500 kD, in preferred embodiments greater than 750kD and most preferred greater than 1000kD. In certain other embodiments, the initial molecular weight of dextran is between 1000kD and 2000kD, in preferred embodiments the initial molecular weight of dextran is approximately 1000-1500kD.
- In certain embodiments, the components of the kit are dry mixed in an amount that, when added to an aqueous solution, yields a gamma stabilized dextran solution of approximately 1 to 10 wt% of dextran in an aqueous solution. Preferably the dextran is 1 to 5wt % and more preferable 2 to 4wt % dextran when used in an aqueous solution.
- In certain embodiments, the kit may further comprise buffers such as phosphate buffered saline (PBS), saline and or other non-toxic enhancers such as, sodium citrate, sodium succinate and combinations thereof. The additives may be combined in such a way that when added together in an aqueous solution, a gamma stabilized dextran solution is obtained. In certain embodiments, the kit is prepared in such a way that the individual components are provided separately. In other embodiments, the kit is prepared such that components in solid form may be premixed and supplied together. In still other embodiments, the kit is prepared such that certain components are provided in solution.
- The methods and kits of the invention to sediment blood cells generally comprise adding the gamma stabilized dextran solution to accelerate RBC sedimentation. For example, in one embodiment, a sample that includes red blood cells is treated by adding an irradiated gamma stabilized dextran solution, followed by incubation of the sample, and eventual recovery of the total nucleated cells (TNCs).
- One or more of the methods of recovering a percentage of TNCs from a sample comprising red blood cells comprises adding a gamma stabilized dextran solution which has been irradiated, at a predetermined concentration,, incubation of the sample, and eventually recovering of the total nucleated cells.
- Practice of the invention will be more fully understood from the following examples, which are presented herein for illustration only and should not be construed as limiting the invention in any way.
- Materials: Human cord blood was used for the experiments. The dextran 1230 used in this example was obtained from GE Healthcare, (Uppsala, Sweden). Ammonium formate and sodium ascorbate are available from Sigma-Aldrich (St. Louis, Missouri). Sodium citrate was obtained from Thermo Fisher Scientific (Waltham, Massachusetts). Pure dextran samples were prepared by dissolving approximately 37.5mM of sodium citrate and 2.25% (w/v) of dextran in 40% (v/v) of saline (Baxter, Deerfield IL) and 31.6 % (v/v) of water for injection (Baxter, IL). Dextran and sodium citrate were allowed to dissolve for 2 hours after which the remaining saline was added to make up the desired volume. All samples were allowed to dissolve for at least 2hr prior to analysis.
- Analysis of dextrans was accomplished using Gel Permeation Chromatography in Combination with Multi-Angle Laser Light Scattering.
- Static Light Scattering analysis in combination with aqueous phase gel permeation chromatography was achieved using an Agilent 1100 series HPLC in combination with a Wyatt Technologies Dawn EOS Multi-Angle Light Scattering Detector in-line to a Wyatt Optilab DSP Interferometric Refractive index detector. The light scattering detector collects scattered light at 18 angles upstream of the refractive index (DRI) data acquisition. The function of the DRI detector is to provide a concentration term from the RI response of a given analyte with a known refractive index increment value (DN/DC) for use in the first principle calculation equation for molar mass. The chromatography was achieved using an isocratic elution of 2mM ammonium formate (pH 4-5). The molar mass values were obtained using the first principle calculation as derived from the Zimm formalism.
- The GPC separation was achieved using 2 Tosoh PW Columns: 1-G6000 and 1-G3000 aqueous SEC columns (7.5x300mm) in series at a flow rate of 1ml min-1 run at ambient temperature as outlined in Table 1.
Table 1. Method Parameters for Gel Permeation Chromatography in combination with Multi-Angle Laser Light Scattering / Differential Refractive Index Detection of Dextrans. Instrument Agilent 1100 Series HPLC / Wyatt EOS Multi- Angle Laser Light Scattering Detector w/ Quasi-Elastic LS Accessory (QELS) / Optilab Differential Refractive Index Detector Column 1-Tosoh G3000 PW (7 x 300mm) and 1-Tosoh G3000 PW (7 x 300mm) Mobile Phase 2mM Ammonium Formate pH=4 -5 Flow 1ml / min Temperature Ambient (28-30C) Injection Volume 20ul DRI Temperature 35C DNDC 0.145 ml/g Gradient Profile Isocratic Elution - Table 2 shows the effects of ascorbic acid or its sodium salt added from 1.0 to 10.0 wt% to an aqueous dextran solution followed by exposure to 40 kGy dose of Gamma irradiation (Cobalt 60)
Table 2. Dextran solutions exposed to 40kGy dose of gamma radiation Sample # Dextran Mw (kD) % Ascorbate Gamma dose (kGy) Mw after irradiation 6 Dextran AB (1,230) 0 none 1,230 12 Dextran AB (1,230) 0 40 35 13 Dextran AB (1,230) 1 40 51 14 Dextran AB (1,230) 2 40 95 13 Dextran AB (1,230) 4 40 342 14 Dextran AB (1,230) 5 40 413 15 Dextran AB (1,230) 6 40 452 16 Dextran AB (1,230) 10 40 614 - As can be seen from data in Table 3, severe Mw drop was noted even at the low 20kGy dose of gamma radiation. However a significant response to added wt% ascorbate versus molecular weight retention was observed notably around 4 wt%.
Table 3. 20 kGy Experimental Results Sample Dextran source Wt% ascorbate Gamma dose (kGy) Mw after irradiation control Dextran AB (1,230) 0 20 21 10 Dextran AB (1,230) 5 20 586 11 Dextran AB (1,230) 6 20 616 - At the lower dose of 20 kGy (Table 3) significantly less ascorbate was required to retain molecular weight.
- Dextran having a Mw of greater than 200 kD is desirable for optimal sedimentation performance. Using combination of starting high molecular weight dextran in the presence of ascorbate achieves that goal. However due to the presence of ascorbate it was still desirable to confirm the cord blood sedimentation performance.
- The extent of red blood cell aggregation was measured in vitro by mixing 5 mls cord blood (sourced from New York Blood Center) in a 20 ml tube (from Globe Scientific) with 10mls of aqueous dextran solutions from Table 4. A control sample was also prepared without the sodium ascorbate. The tube was capped and the contents mixed well by inverting the tube up and down. The contents were allowed to settle under gravity and the height of the RBC pellet was measured at 0,10,20,40, and 60 minutes.
- As shown in Table 4 a number of the above samples were added to cord blood to assess the RBC sedimentation performance.
Table 4. Sedimentation Performance Sample # Material % Ascorbate Gamma Dose (kGy) MW after irradiation Aggregation (cm) 0 min 20 min 60 min 1 Dextran 0 none 1,230 12.2 3.0 2.4 2 Dextran 4 none 1,230 11.9 2.6 2.0 3 Dextran 0 40 35 12.0 11.8 11.5 4 Dextran 4 40 342 12.2 3.2 2.1 5 Dextran 5 40 413 12.0 3.5 2.0 6 Dextran 6 40 452 12.0 4.4 2.1 - Table 4 shows that gamma irradiated dextran's in the presence of sodium ascorbate aggregates RBCs to similar extents as non-irradiated dextran's with and without sodium ascorbate.
Claims (23)
- An aqueous dextran solution, stable to gamma irradiation comprising:1 to 10 wt /v % of dextran where the dextran has an initial average molecular weight greater than 500kD;2.0 to 20.0 wt% ascorbic acid or its mineral salt to the dextran.
- The solution of claim 1 where the dextran has an initial molecular weight greater than 750kD.
- The solution of claim 2 where the dextran has an initial molecular weight between approximately 1000 to 1500kD.
- The solution of claim 1 where the mineral salt is sodium ascorbate.
- The solution of claim 1 further comprising a buffer, a non-toxic enhancer or a combination thereof.
- The solution of claim 5 where the non-toxic enhancer is sodium citrate, sodium succinate, or a combination thereof.
- The solution of claim 5 where the buffer comprises organic or inorganic salts that maintain a pH of 4.0 to 8.0.
- A kit for producing a gamma stabilized aqueous dextran solution comprising:dextran where the dextran has an initial average molecular weight greater than 500kD; and2.0 to 20.0 wt% ascorbic acid or its mineral salt to the dextran.
- The kit of claim 8 where the dextran initial molecular weight is greater than 750kD.
- The kit of claim 9 where the dextran initial molecular weight is between approximately 1000 to 1500kD.
- The kit of claim 8 where the mineral salt is sodium asorbate.
- The kit of claim 8 further comprising a buffer, a non-toxic enhancer or a combination thereof.
- The kit of claim 12 where the non-toxic enhancer is sodium citrate, sodium succinate, or a combination thereof.
- The kit of claim 12 where the buffer comprises organic or inorganic salts that maintain a pH of 4.0-9.0.
- A method to aggregate cells in a sample comprising red blood cells (RBC), comprising the steps of:a. obtaining an aqueous dextran solution, the aqueous dextran solution comprising;1 to 10 wt/v % of dextran where the dextran has an initial average molecular weight greater than 500kD; and2.0 to 20.0 wt% ascorbic acid or its mineral salt to the dextran;b. exposing the solution to gamma radiation at a dose between 20 and 50 kGy; andc. adding the sample to the aqueous dextran solution.
- The method of claim 15 where the dextran has an initial molecular weight greater than 750kD.
- The method of claim 16 where the dextran has an initial molecular weight between approximately 1000 to 1500kD.
- The method of claim 15 where the mineral salt is sodium asorbate.
- The method of claim 15 where the aqueous solution further comprises a buffer, a non-toxic enhancer or a combination thereof.
- The method of claim 19 where the non-toxic enhancer is sodium citrate, sodium succinate, or a combination thereof.
- The method of claim 19 where the buffer comprises organic or inorganic salts that maintain a pH of 4.0 to 8.0.
- The method of claim 15 further comprising the steps of incubating the sample to aggregate and sediment of the red blood cells and optionally recovering total nucleated cells (TNC) from the sample.
- The method of claim 22 were recovering the TNC comprises concentration of a liquid phase, said liquid phase comprising plasma and dextran using centrifugation, membrane filtration, or a combination thereof.
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US20030109494A1 (en) * | 2001-12-08 | 2003-06-12 | Hebert Rolland F. | Ascorbic acid stability |
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